Method of enhancing proliferation and/or hematopoietic differentiation of stem cells

ABSTRACT

The present invention provides a method for enhancing the proliferation and/or hematopoietic differentiation and/or maintenance of mammalian stem cells. The method is useful for generating expanded populations of hematopoietic stem cells (HSCs) and thus mature blood cell lineages. This is desirable where a mammal has suffered a decrease in hematopoietic or mature blood cells as a consequence of disease, radiation or chemotherapy. The method of the present invention comprises increasing the intracellular level of a cdx in stem cells, including hematopoietic stem cells, in culture, either by providing an exogenous cdx protein to the cell, or by introduction into the cell of a genetic construct encoding a cdx. The cdx is selected from the cdx family and includes cdx1, cdx2, or cdx4. The cdx may be a wild type protein appropriate for the species from which the cells are derived, or a mutant form of the protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation application of co-pending U.S. patentapplication Ser. No. 12/191,402 filed Aug. 14, 2008, which is aContinuation application of U.S. patent application Ser. No. 10/528,808filed Mar. 23, 2005 now issued as U.S. Pat. No. 7,427,603 issued on Sep.23, 2008, which is U.S. National Stage Entry under 35 U.S.C. §371 ofInternational Application No. PCT/US03/29185, filed Sep. 18, 2003, whichclaims priority from U.S. Provisional Application No. 60/413,816, filedon Sep. 26, 2002.

BACKGROUND OF THE INVENTION

Chemo- and radiation therapies cause dramatic reductions in blood cellpopulations in cancer patients. At least 500,000 cancer patients undergochemotherapy and radiation therapy in the US and Europe each year andanother 200,000 in Japan. Bone marrow transplantation therapy of valuein aplastic anemia, primary immunodeficiency and acute leukemia(following total body irradiation) is becoming more widely practiced bythe medical community. At least 15,000 Americans have bone marrowtransplants each year. Other diseases can cause a reduction in entire orselected blood cell lineages. Examples of these conditions includeanemia (including macrocytic and aplastic anemia); thrombocytopenia;hypoplasia; immune (autoimmune) thrombocytopenic purpura (ITP); and HIVinduced ITP.

Pharmaceutical products are needed which are able to enhancereconstitution of blood cell populations of these patients.

SUMMARY OF THE INVENTION

The present invention provides a method for enhancing the proliferationand/or hematopoietic differentiation and/or maintenance of mammalianstem cells. The method is useful for generating expanded populations ofhematopoietic stem cells (HSCs) and thus mature blood cell lineages.This is desirable where a mammal has suffered a decrease inhematopoietic or mature blood cells as a consequence of disease,radiation or chemotherapy. The method of the present invention comprisesincreasing the intracellular level of a cdx in stem cells, includinghematopoietic stem cells, in culture, either by providing an exogenouscdx protein to the cell, or by introduction into the cell of a geneticconstruct encoding a cdx. The cdx is selected from the cdx family andincludes cdx1, cdx2, or cdx4. The cdx may be a wild type proteinappropriate for the species from which the cells are derived, or amutant form of the protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-5 show that cdx4 alters hox gene expression in zebrafish andmouse cells and induces blood development in embryoid bodies.

FIG. 1 shows effect of cdx4 and HoxB4 overexpression on hematopoieticprogenitors derived from embryoid bodies (EBs). Colony forming unitsscored are macrophage (Mac), megakaryocytes and mixed lineage (Meg-mix),granulocyte, macrophage (GM), and granulocyte, macrophage, megakaryocyte(GEMM). Photographs of representative colonies are shown below thegraph.

FIG. 2 shows quantitative PCR analysis of the expression of selectedHoxA, HoxB, and HoxC cluster genes in EBs overexpressing cdx4.

FIG. 3 shows RT-PCR analysis of cdx4 expression during EB development.

FIG. 4 shows the effect of cdx4 overexpression on hematopoieticdevelopment during different stages of EB development usingtetracycline-inducible murine embryonic stem cell lines. cdx4 expressionwas induced by the addition of doxycycline between the days indicatedbelow the graph and hematopoietic colony formation was assayed at day 6.The types of colonies scored were the same as above, which the additionof primitive and definitive erythroid colonies (Ery-P and Ery-D,respectively) and mast cell colonies (Mast).

FIG. 5 shows a model for the role of cdx4 in AP patterning and blooddevelopment. Signaling molecules such as FGFs, Wnts, and retinoic acid(RA) are known to regulate the expression of cdx4, which in turnestablishes the correct expression domains of hox genes necessary forblood development. In the absence of cdx4 (right panel), hox expressiondomains are shifted and fewer erythroid cells are formed.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for enhancing the proliferationand/or hematopoietic differentiation and/or maintenance of mammalianstem cells. The method is useful for generating expanded populations ofhematopoietic stem cells (HSCs) and thus mature blood cell lineages.This is desirable where a mammal has suffered a decrease inhematopoietic or mature blood cells as a consequence of disease,radiation, chemotherapy or congenital anemia (e.g., Diamond BlackfanAnemia). The method of the present invention comprises increasing theintracellular level of a cdx in stem cells, including hematopoietic stemcells, in culture, either by providing an exogenous cdx protein to thecell, or by introduction into the cell of a genetic construct encoding acdx. The cdx is selected from the cdx family and includes cdx1, cdx2, orcdx4. The cdx may be a wild type protein appropriate for the speciesfrom which the cells are derived, or a mutant form of the protein.

The differentiated and expanded cell populations are useful as a sourceof hematopoietic stem cells, which may be used in transplantation torestore hematopoietic function to autologous or allogeneic recipients.

In one embodiment, mammalian stem cells are differentiated to HSCs invitro by increasing the level of cdx in the cell. In another embodiment,the number of HSCs in a culture is expanded by increasing the levels ofcdx in the cell. The intracellular levels of cdx may be manipulated byproviding exogenous cdx protein to the cell, or by introduction into thecell of a genetic construct encoding a cdx. The cdx may be a wild-typeor a mutant form of the protein.

The term cdx, as used herein, is intended to refer to both wild-type andmutant forms of the cdx protein family, and to fusion proteins andderivatives thereof. Usually the protein will be of mammalian origin,although the protein from other species may find use. The sequences ofmany cdx proteins are publicly known. Preferably, the mammal is a humanand the cdx is selected from the group consisting of cdx1(GenBankaccession number NM_(—)001804; Suh et al., J. Biol. Chem. 277:35795(2002)), cdx2 (GenBank accession number NM_(—)001265; Yamamoto et al.,Biochem. Biophys. Res. Commun. 300(4):813 (2003)), or cdx4 (GenBankaccession number NM_(—)005193; Horn et al., Hum. Mol. Genet. 4(6),1041-1047 (1995)).

In one embodiment of the invention, the cdx is delivered to the targetedstem cells by introduction of an exogenous nucleic acid expressionvector into the cells. Many vectors useful for transferring exogenousgenes into target mammalian cells are available. The vectors may beepisomal, e.g. plasmids, virus derived vectors such cytomegalovirus,adenovirus, etc., or may be integrated into the target cell genome,through homologous recombination or random integration, e.g. retrovirusderived vectors such MMLV, HIV-1, ALV, etc.

Retrovirus based vectors have been shown to be particularly useful whenthe target cells are hematopoietic stem cells. For example, see Baum etal. (1996) J Hematother 5(4):323-9; Schwarzenberger et al. (1996) Blood87:472-478; Nolta et al. (1996) P.N.A.S. 93:2414-2419; and Maze et al.(1996) P.N.A.S. 93:206-210. Lentivirus vectors have also been describedfor use with hematopoietic stem cells, for example see Mochizuki et al.(1998) J Virol 72(11):8873-83. The use of adenovirus based vectors withhematopoietic cells has also been published, see Ogniben and Haas (1998)Recent Results Cancer Res 144:86-92.

Various techniques known in the art may be used to transfect the targetcells, e.g. electroporation, calcium precipitated DNA, fusion,transfection, lipofection and the like. The particular manner in whichthe DNA is introduced is not critical to the practice of the invention.

Combinations of retroviruses and an appropriate packaging line may beused, where the capsid proteins will be functional for infecting thetarget cells. Usually, the cells and virus will be incubated for atleast about 24 hours in the culture medium. Commonly used retroviralvectors are “defective”, i.e. unable to produce viral proteins requiredfor productive infection. Replication of the vector requires growth inthe packaging cell line.

The host cell specificity of the retrovirus is determined by theenvelope protein, env (p120). The envelope protein is provided by thepackaging cell line. Envelope proteins are of at least three types,ecotropic, amphotropic and xenotropic. Retroviruses packaged withecotropic envelope protein, e.g. MMLV, are capable of infecting mostmurine and rat cell types. Ecotropic packaging cell lines include BOSC23(Pear et al. (1993) P.N.A.S. 90:8392-8396). Retroviruses bearingamphotropic envelope protein, e.g. 4070A (Danos et al, supra.), arecapable of infecting most mammalian cell types, including human, dog andmouse. Amphotropic packaging cell lines include PA12 (Miller et al.(1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller et al. (1986) Mol.Cell. Biol. 6:2895-2902) GRIP (Danos et al. (1988) PNAS 85:6460-6464).Retroviruses packaged with xenotropic envelope protein, e.g. AKR env,are capable of infecting most mammalian cell types, except murine cells.

The sequences at the 5′ and 3′ termini of the retrovirus are longterminal repeats (LTR). A number of LTR sequences are known in the artand may be used, including the MMLV-LTR; HIV-LTR; AKR-LTR; FIV-LTR;ALV-LTR; etc. Specific sequences may be accessed through publicdatabases. Various modifications of the native LTR sequences are alsoknown. The 5′ LTR acts as a strong promoter, driving transcription ofthe cdx gene after integration into a target cell genome. For some uses,however, it is desirable to have a regulatable promoter drivingexpression. Where such a promoter is included, the promoter function ofthe LTR will be inactivated. This is accomplished by a deletion of theU3 region in the 3′ LTR, including the enhancer repeats and promoter,that is sufficient to inactivate the promoter function. Afterintegration into a target cell genome, there is a rearrangement of the5′ and 3′ LTR, resulting in a transcriptionally defective provirus,termed a “self-inactivating vector”.

Suitable inducible promoters are activated in a desired target celltype, either the transfected cell, or progeny thereof. Bytranscriptional activation, it is intended that transcription will beincreased above basal levels in the target cell by at least about 100fold, more usually by at least about 1000 fold. Various promoters areknown that are induced in hematopoietic cell types, e.g. IL-2 promoterin T cells, immunoglobulin promoter in B cells, etc.

In an alternative method, expression vectors that provide for thetransient expression in mammalian cells may be used. In general,transient expression involves the use of an expression vector that isable to replicate efficiently in a host cell, such that the host cellaccumulates many copies of the expression vector and, in turn,synthesizes high levels of a desired polypeptide encoded by theexpression vector. Transient expression systems, comprising a suitableexpression vector and a host cell, allow for the convenient short termexpansion of cells, but do not affect the long term genotype of thecell.

In some cases it may be desirable to provide exogenous cdx protein,rather than transducing the cells with an expression construct. The cdxprotein may be added to the culture medium at high levels. Preferablythe cdx protein is modified so as to increase its transport into thecells. See, for example, US 2002/0086383.

In one embodiment of the invention, tat protein is used to deliver cdx.The preferred transport polypeptides are characterized by the presenceof the tat basic region amino acid sequence (amino acids 49-57 ofnaturally-occurring tat protein); the absence of the tat cysteine-richregion amino acid sequence (amino acids 22-36 of naturally-occurring tatprotein) and the absence of the tat exon 2-encoded carboxy-terminaldomain (amino acids 73-86 of naturally-occurring tat protein). Transportpolypeptides are attached to cdx by chemical cross-linking or by geneticfusion, where the cdx moiety may be a wild-type or stabilized form. Aunique terminal cysteine residue is a preferred means of chemicalcross-linking.

The term stem cell is used herein to refer to a mammalian cell that hasthe ability both to self-renew, and to generate differentiated progeny(see Morrison et al. (1997) Cell 88:287-298). Generally, stem cells alsohave one or more of the following properties: an ability to undergoasynchronous, or symmetric replication, that is where the two daughtercells after division can have different phenotypes; extensiveself-renewal capacity; capacity for existence in a mitotically quiescentform; and clonal regeneration of all the tissue in which they exist, forexample the ability of hematopoietic stem cells to reconstitute allhematopoietic lineages. “Progenitor cells” differ from stem cells inthat they typically do not have the extensive self-renewal capacity, andoften can only regenerate a subset of the lineages in the tissue fromwhich they derive, for example only lymphoid, or erythroid lineages in ahematopoietic setting.

Stem cells may be characterized by both the presence of markersassociated with specific epitopes identified by antibodies and theabsence of certain markers as identified by the lack of binding ofspecific antibodies. Stem cells may also be identified by functionalassays both in vitro and in vivo, particularly assays relating to theability of stem cells to give rise to multiple differentiated progeny.

Stem cells can be derived from a human donor, e.g., pluripotenthematopoietic stem cells, adult somatic stem cells, and the like.Embryonic stem cells may also be used. Stem cells can also be obtainedfrom umbilical cord blood, amniotic fluid, chorionic villus andplacenta. See, WO03042405.

Other hematopoietic “progenitor” cells of interest include cellsdedicated to lymphoid lineages, e.g. immature T cell and B cellpopulations. The methods of the present invention are useful inexpanding selected populations of these cells.

Purified populations of stem or progenitor cells may be used to initiatethe cultures. For example, human hematopoietic stem cells may bepositively selected using antibodies specific for CD34, thy-1; ornegatively selected using lineage specific markers which may includeglycophorin A, CD3, CD24, CD16, CD14, CD38, CD45RA, CD36, CD2, CD19,CD56, CD66a, and CD66b.

The cells of interest are typically mammalian, where the term refers toany animal classified as a mammal, including humans, domestic and farmanimals, and zoo, laboratory, sports, or pet animals, such as dogs,horses, cats, cows, mice, rats, rabbits, etc. Preferably, the mammal ishuman.

The cells which are employed may be fresh, frozen, or have been subjectto prior culture. They may be fetal, neonate, adult. Hematopoietic cellsmay be obtained from fetal liver, bone marrow, blood, particularly G-CSFor GM-CSF mobilized peripheral blood, cord blood or any otherconventional source. The manner in which the stem cells are separatedfrom other cells of the hematopoietic or other lineage is not criticalto this invention. As described above, a substantially homogeneouspopulation of stem or progenitor cells may be obtained by selectiveisolation of cells free of markers associated with differentiated cells,while displaying epitopic characteristics associated with the stemcells.

The stem or progenitor cells are grown in vitro in an appropriate liquidnutrient medium. Generally, the seeding level will be at least about 10cells/ml, more usually at least about 100 cells/ml and generally notmore than about 10⁵ cells/ml, usually not more than about 10⁴ cells/ml.

Various media are commercially available and may be used, including Exvivo serum free medium; Dulbecco's Modified Eagle Medium (DMEM), RPMI,Iscove's medium, etc. The medium may be supplemented with serum or withdefined additives. Appropriate antibiotics to prevent bacterial growthand other additives, such as pyruvate (0.1-5 mM), glutamine (0.5-5 mM),2-mercaptoethanol may also be included.

Culture in serum-free medium is of particular interest. The medium maybe any conventional culture medium, generally supplemented withadditives such as iron-saturated transferrin, human serum albumin, soybean lipids, linoleic acid, cholesterol, alpha thioglycerol, crystallinebovine hemin, etc., that allow for the growth of hematopoietic cells.

Preferably the expansion medium is free of cytokines, particularlycytokines that induce cellular differentiation. The term cytokine mayinclude lymphokines, monokines and growth factors. Included among thecytokines are thrombopoietin (TPO); nerve growth factors;platelet-growth factor; transforming growth factors (TGFs);erythropoietin (EPO); interferons such as interferon-α, β, and γ; colonystimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1γ, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-11, IL-12; etc. In some circumstances,proliferative factors that do not induce cellular differentiation may beincluded in the cultures, e.g. c-kit ligand, LIF, and the like.

Frequently stem cells are isolated from biological sources in aquiescent state. Certain expression vectors, particularly retroviralvectors, do not effectively infect non-cycling cells. Culturesestablished with these vectors as a source of cdx sequences are inducedto enter the cell cycle by a short period of time in culture with growthfactors. For example, hematopoietic stem cells are induced to divide byculture with c-kit ligand, which may be combined with LIF, IL-11 andthrombopoietin. After 24 to 72 hours in culture with cytokines, themedium is changed, and the cells are exposed to the retroviral culture,using culture conditions as described above.

After seeding the culture medium, the culture medium is maintained underconventional conditions for growth of mammalian cells, generally about37° C. and 5% CO₂ in 100% humidified atmosphere. Fresh media may beconveniently replaced, in part, by removing a portion of the media andreplacing it with fresh media. Various commercially available systemshave been developed for the growth of mammalian cells to provide forremoval of adverse metabolic products, replenishment of nutrients, andmaintenance of oxygen. By employing these systems, the medium may bemaintained as a continuous medium, so that the concentrations of thevarious ingredients are maintained relatively constant or within apre-described range. Such systems can provide for enhanced maintenanceand growth of the subject cells using the designated media andadditives.

These cells may find various applications for a wide variety ofpurposes. The cell populations may be used for screening variousadditives for their effect on growth and the mature differentiation ofthe cells. In this manner, compounds which are complementary, agonistic,antagonistic or inactive may be screened, determining the effect of thecompound in relationship with one or more of the different cytokines.

The populations may be employed as grafts for transplantation. Forexample, hematopoietic cells are used to treat malignancies, bone marrowfailure states and congenital metabolic, immunologic and hematologicdisorders. Marrow samples may be taken from patients with cancer, andenriched populations of hematopoietic stem cells isolated by means ofdensity centrifugation, counterflow centrifugal elutriation, monoclonalantibody labeling and fluorescence activated cell sorting. The stemcells in this cell population are then expanded in vitro and can serveas a graft for autologous marrow transplantation. The graft will beinfused after the patient has received curative chemo-radiotherapy.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

EXAMPLES Introduction

The formation of blood cells during vertebrate development occurs insuccessive stages in anatomically distinct sites⁶. In amniotes, thefirst wave (known as primitive or embryonic hematopoiesis) originates inthe yolk sac blood islands and is characterized by the formation oferythroid and endothelial cells. The coincident onset of bothhematopoiesis and vasculogenesis in the yolk sac has led to thehypothesis that both cell types are derived from a common precursor,termed the hemangioblast⁷. In zebrafish, embryonic hematopoiesis occursin an intra-embryonic location known as the intermediate cell mass(ICM). The ICM develops along the trunk midline by the convergence ofbilateral stripes of hematopoietic and vascular precursors. One of theearliest molecular markers of these ICM precursors is the stem cellleukemia (scl) gene, which encodes a basic helix-loop-helixtranscription factor^(8, 9). Gene targeting studies in mice havedemonstrated that scl is necessary for the development of allhematopoietic lineages. In contrast, endothelial cells are present inscl null embryos but fail to remodel properly in the yolk sac^(10, 11).Studies in zebrafish have shown that overexpression of scl duringdevelopment is sufficient to induce ectopic blood and vascular cells andthese findings have led to the suggestion that scl is capable ofspecifying hemangioblast fate from mesoderm⁸.

While fate-mapping studies in zebrafish have shown that embryonic bloodcells arise from ventral mesoderm of the late blastula^(12, 13) themolecular pathways responsible for inducing the early expression of sclare largely unknown. In general, posterior tissues of mesodermal originare derived from ventral mesoderm whereas anterior tissues descend frommore dorsal mesoderm. Consistent with this, genes that ‘ventralize’ theearly gastrula embryo, such as the bone morphogenetic proteins (BMPs),induce an expansion of blood and posterior tissues at the expense ofmore anterior structures such as the head¹⁴. Thus, factors thatdetermine posterior cell fates along the anteroposterior (AP) axis mustalso be intimately connected with genes that specify ventral fatesincluding blood.

The establishment of tissues along the AP axis of the embryo isdependent upon the homeobox transcription factors encoded by the hoxgenes¹⁵. Within the genome, these genes are grouped together in clusters(HoxA, HoxB, HoxC and HoxD) and are expressed in overlapping domainsalong the AP axis with their anterior expression limits correlating totheir physical order within the cluster. Perturbations in these anteriorexpression boundaries result in changes in cell fate and this has led tothe ‘Hox code’ hypothesis, in which specific combinations of Hox genesare believed to specify tissue identities along the AP axis¹⁵. Despitebeing held as critical regulators of embryonic patterning, the effectsof germline disruptions of Hox genes in the mouse are largely restrictedto the axial skeleton, neural crest, central nervous system, andlimbs^(3, 15). The relatively mild phenotypes of single Hox geneknockouts in mice can be explained by extensive functional redundancybetween paralogous genes within each cluster.

A number of studies have demonstrated that ectopically expressed hoxgenes can influence hematopoietic lineage decisions^(4, 5). For example,overexpression of HoxA9, HoxB4, and HoxB7 has been shown to modulate theproliferation/self-renewal of mouse hematopoietic stem cells¹⁶⁻¹⁹. Inaddition, ectopically expressed HoxB4 can induce embryonic hematopoieticprogenitors to acquire properties characteristic of adult hematopoieticstem cells²⁰. Deregulated Hox gene expression is also associated withleukemia transformation^(4, 5). Overexpression of HoxA9^(18, 21) orHoxA10²² in murine bone marrow ultimately leads to acute myeloidleukemia (AML) whereas proviral activation of HoxA7 has been implicatedin myeloid leukaemia²³. A subset of human AML is associated with afusion of the NUP98 gene, which encodes a component of the nuclear porecomplex, to a number of different HOX genes including HOXA9²⁴.Translocations involving the MLL gene, a homologue of MLL that isrequired for the maintenance of HOX gene expression, have also beenimplicated in certain human leukemias²⁵.

In this study we have characterized the zebrafish kugelig (kgg) mutant,which exhibits reduced scl expression, severe anemia, and a shortened APaxis. We identify the kgg locus as the caudal-related homeobox gene cdx4and show that the defect in erythropoiesis is associated with aberranthox gene expression. Overexpressing scl in kgg mutants fails to rescueblood development indicating that the specification of hematopoeiticcell fate is dependent upon cdx4 function. In contrast, erythropoiesisin kgg mutants can be robustly rescued by overexpressing hoxb7a andhoxa9a but not hoxb8a, suggesting that the hematopoietic defects resultdirectly from perturbations in hox gene expression. Overexpression ofcdx4 during zebrafish development or in mouse embryonic stem cellsinduces blood formation and alters hox gene expression patterns. Takentogether, our findings demonstrate that cdx4 is both necessary andsufficient for the formation of embryonic blood cells during vertebratedevelopment.

Example 1 Methods

Computer analysis. The genetic map position of kgg was obtained from theMax-Planck-Institut fur Entwicklungsbiologie (Tübingen, Germany)website. Genomic sequence of the cdx4 locus was obtained from theWellcome Trust Sanger Institute website. RH mapping data was provided bythe Children's Hospital Genome Initiative (Boston, Mass.) website.Protein sequence prediction and alignment were performed using DNAstarsoftware.

Deletion analysis and genotyping. The following primers to each exon ofthe cdx4 gene were used to determine the extent of the kgg^(tv205)deletion by PCR: exon one (forward 5′-AGCTCCTTTTGGACTATTAC-3′ (SEQ IDNO: 1); reverse 5′-CCAACGTACATGATTTGGAA-3′ (SEQ ID NO: 2)), exon two(forward 5′-ATACCTTTTGGAGAAAGAGG-3′ (SEQ ID NO: 3); reverse5′-CCGGTTGATGACGACTGGAC-3′ (SEQ ID NO: 4)), exon three (forward5′-CAAAACGAGAACGAAGGAGA-3′ (SEQ ID NO: 5); reverse5′-ACCTGTCTCTCTGAAAGCCC-3′ (SEQ ID NO: 6)), and exon four (forward5′-TAAGATCTGGTTTCAGAACC-3′ (SEQ ID NO: 7); reverse5′-TGGATGATCCAAGTTCGAGT-3′ (SEQ ID NO: 8)). Exon three forward and exonfour reverse primers were used to genotype kgg^(tv205) embryos. Primersspecific to the ESTs fj63c09, fb79h04, flk1 (fk52c05), fb75e05, chic1(fj33g02), fc54b04, and fi30c11 were obtained from the WashU ZebrafishGenome Resources Project website, while primer sequences for the markersz20545 and z11437 were obtained from the Massachusetts General HospitalZebrafish Server website. 3′ rapid amplification of cDNA ends (RACE) wasperformed using the SMART RACE kit (Clontech), cDNA prepared from 14-15somite stage kgg^(tv205) mutants, and the cdx4-specific primer5′-AGCCTCGGACCTCCAAATTC-3′ (SEQ ID NO: 9). PCR products were subclonedin the pGEM-T easy vector (Promega, Madison, Wis.) and sequenced.

Electrophoretic mobility shift assays. EMSAs were performed using theGel Shift Assay System (Promega, Madison, Wis.) and in vitro translated(IVT) proteins prepared using the TNT SP6 Quick CoupledTranscription/Translation System (Promega, Madison, Wis.).Double-stranded oligonucleotide probes contained a single consensus cdxbinding site (5′-GAGAAATTTATATTGT-3′ (SEQ ID NO: 10); binding siteconsensus is underlined) or mutated site (5′-GAGAAATCCATATTGT-3′ (SEQ IDNO: 11); mutated nucleotides are underlined).³⁵ S-methionine-labelledIVT cdx4 (wt) and the F(170)L mutant proteins were resolved on a 10-20%Tris-HCl polyacrylamide gel (Ready Gels, Biorad, Hercules, Calif.)alongside prestained broad range standards (Biorad) and analyzed byautoradiography.

Fish strains. The kgg^(tv205) and kgg^(tl240) mutant lines were obtainedfrom the Tübingen stock center (Tübingen, Germany) and exhibit a similarseverity of phenotype. Wild-type strains were AB, Tü, and WIK. Fishmaintenance, breeding, and embryo staging were performed according tostandard procedures.

Inducible cdx4 ES cell lines and colony assays. The induciblecdx4-targeting plasmid (plox-cdx4) was generated by subcloning mousecdx4 into the EcoRI/XbaI site of the plox vector²⁰. To make thetetracycline-inducible cdx4 ES cell line, Ainv15 ES cells wereelectroporated with 20 ug of plox-cdx4 and 20 ug of pSalk-Cre, followedby selection with G418 (400 ug/ml) in ES culture medium. Coloniespositive for plox-cdx4 were confirmed by RT-PCR. Thetetracycline-inducible cdx4 ES cells and EBs were maintained andproduced as described previously²⁰. Briefly, day 2 EBs fromhanging-drops were harvested and cultured in rotating Petri dishes.Doxycycline was added into EB medium for the indicated time periods andthen removed by three washes of PBS, followed by ES culture medium. EBswere collected at day 6 by collagenase treatment and plated intoMethocult GF M3434 (StemCell Technologies). The colonies were scored 6-9days later.

Microinjection. Wild-type and F(170)L mutant cdx4 cDNAs were subclonedinto the expression vector pCS2⁺, linearized with NotI, and syntheticmRNA made using the mMessage mMachine kit (Ambion, Austin, Tex.). CappedRNA was resuspended in sterile water and 500 pl was injected between theone- to four-cell stages at a concentration of 30 ng/μl. Full-lengthhoxb6b, hoxb7a hoxb8a, and hoxa9a were amplified from 5-somite stagecDNA by RT-PCR using forward (hoxb6b:5′-ATGCGAATTCCCCATGAGTTCCTATTTCGTCA-3′ (SEQ ID NO: 12); hoxb7a:5′-ATGCGAATTCACCATGAGTTCATTGTATTATGCG-3′ (SEQ ID NO: 13); hoxb8a:5′-ATGCGAATTCACCATGAGCTCATATTTCGTCAAC-3′ (SEQ ID NO: 14); hoxa9a:5′-ATGCGAATTCACCATGTCGACATCCGGAGCT-3′ (SEQ ID NO: 15); start codonunderlined)) and reverse (hoxb6b: 5′-GCATCTCGAGCTACATTCTACATGTTATGTAC-3′(SEQ ID NO: 16); hoxb7a: 5′-GACTCTCGAGCTACTCATCATCTTCTTCTTC-3′ (SEQ IDNO: 17); hoxb8a: 5′-GCATCTCGAGCTACATTTGTTTTGCCTTGTC-3′ (SEQ ID NO: 18);hoxa9a: 5′-GATCTCTAGATTAGTCTTCCTTCGTTTC-3′ (SEQ ID NO: 19); stop codonunderlined) primers and subcloned (along with scl) into pCS2+. SyntheticmRNAs were prepared as above and 500 pl was injected at a concentrationof 200, 6 and 2-4 ng/μl, for scl, hoxb7a/hoxa9a, and hoxb6b/hoxb8a,respectively. The cdx4 morpholinos (CGTACATGATTTGGAAGAAACCCCT (SEQ IDNO: 20); start codon underlined) were obtained from Gene Tools LLC(Corvallis, Oreg.) and solubilized in 1× Danieau solution (58 mM NaCl,0.7 mM KCl, 0.4 mM MgSO4, 5 mM HEPES, pH 7.6) at a stock concentrationof 35 mg/ml. One- to four-cell stage embryos were injected with 1 nl ofcdx4 morpholino or an unrelated control morpholino (provided by GeneTools LLC) at a concentration of 0.2 mg/ml. Injections were performed ona PLI-100 microinjector (Medical systems corp., NY).

Mutation analysis by RT-PCR. Total RNA was prepared from kgg^(tv205) andkgg^(tl240) mutant and wild-type embryos at 24 h.p.f. using establishedprocedures and reverse transcribed using Superscript II RNAse H-reversetranscriptase (Invitrogen, Carlsbad, Calif.). The cdx4 ORF was amplifiedusing forward (5′-CATGTACGTTGGATACCTTTTGG-3′ (SEQ ID NO: 21)) andreverse (5′-TCCACAACCCACGCCTCTTATT-3′ (SEQ ID NO: 22)) primers,subcloned into the pGEM-T easy vector (Promega, Madison, Wis.) andsequenced. Our cDNA sequence of wild-type cdx4 differs from thepublished sequence (Genbank accession number NM_(—)131109) by theaddition of two cytosine nucleotides at +709-710. These extranucleotides are also found in the cdx4 genomic sequence deposited in theSanger Center database. The resulting frameshift changes the openreading frame of the carboxy terminus to give a predicted protein of 271residues rather than the published length of 301 resides³². The F(170)Lmutation of the kgg^(tl240) allele was confirmed by sequencing sixindependent clones.

Radiation hybrid mapping. The cdx4 gene was mapped onto the GoodfellowRH panel by the Children's Hospital Genome Initiative group (Boston,Mass.) using the following forward (5′-AGGCGTGGGTTGTGGATTAC-3′ (SEQ IDNO: 23)) and reverse (5′-GATACACTCACCACATACAG-3′ (SEQ ID NO: 24))primers. The contig encoding the foreign exon spliced onto exon 2 ofcdx4 in kgg^(tl240) mutants was mapped using forward(5′-GTGATCAACAACACGTCC-3′ (SEQ ID NO: 25)) and reverse primers(5′-GGAATCTCCTGTCAGCTG-3′ (SEQ ID NO: 26)).

Retroviral expression of cdx4 in ES cells and quantitative PCR. Murinecdx4 was subcloned into the retroviral expression vector MSCV-IRES-GFP(pMIG) and retroviruses were generated using an ecotropic packagingvector and co-transfection to make viral supernatents. Embryoid bodieswere formed from wild-type (RW4) ES cells by differentiating for 6 daysand then definitive hematopoietic cells were enriched using an anti-CD41magnetic strategy resulting in a 10-fold enrichment of CD41/c-Kit⁺cells. Approximately one million enriched cells were plated on OP9monolayers in a 6-well dish and subjected to two rounds of retroviralinfection with either GFP only or cdx4/GFP retroviral supernatants.After 48 hours, GFP cells were sorted and were either directly lysed inTrizol (Invitrogen, Carlsbad, Calif.) for RNA preparation, or wereplated in methylcellulose (M3434, StemCell Technologies) and scored forcolony types 3-7 days later. Representative colonies were cytospun andstained using Jorvet J-322 Dip Quik, (Jorgensen Laboratories Inc.,Loveland, Colo.). To quantitate the relative level of Hox gene mRNA,random hexamer-primed cDNA was prepared from total RNA from either GFPexpressing or cdx4/GFP-expressing cells. Real time PCR measurements wereperformed with an ABI Prism 7700 Sequence Detector and dual labeledprobes (sequence available on request), with the exception of HoxB4,which was quantitated using Sybr green reagents (Applied Biosystems).PCR reactions were performed in triplicate with internal references(GAPDH) used to normalize samples. Hox expression levels are expressedin arbitrary units (relative to the lowest sample) using the comparativeC_(T) method.

In situ hybridization and sectioning. In situ hybridization of mouseembryos was performed as previously described⁵⁶. Whole mount In situhybridization of zebrafish embryos was performed with double stainingusing the red substrate BCIP-INT. Embryos were fixed overnight in 4%paraformaldehyde, transferred to glycerol, flat-mounted under glasscoverslips when possible, and photographed. The following riboprobeswere used: cdx4, cxcr4, flk1, fli1, gata1, globin e3, hoxb5a, hoxb6b,hoxb7a, hoxb8a, hoxa9a, myoD, par1, pax2.1, runx1, scl, and wt1.Full-length cDNAs of the following hox genes were isolated by RT-PCRfrom 5-somite stage cDNA and subcloned into pCS2+ for riboprobesynthesis: hoxb4 (forward 5′-ATGCGAATTCACCATGGCCATGAGTTCCTATTTG-3′ (SEQID NO: 27); reverse 5′-GCATCTCGAGCTATAGACTTGGCGGAGGTCC-3′ (SEQ ID NO:28)), hoxb8b (forward 5′-ATGCGAATTCACCATGAGTTCCTACTTCGTCAAT-3′ (SEQ IDNO: 29); reverse 5′-GCATCTCGAGCTATTTAGAATTGCTAGAAGC-3′ (SEQ ID NO: 30)).Embryos to be sectioned were infiltrated in JB-4 resin, cut at athickness of 5 μm, and then counterstained in 0.5% Safranin O beforebeing mounted.

Results Characterization of the kgg Mutant

We found that embryos homozygous for kugelig (kgg), an autosomalrecessive mutation that was initially identified due to tail defects²⁶,exhibit severe anemia within the first day of development. Two kggalleles, kgg^(tv205) and kgg^(tl240), of equal severity have beenisolated²⁶. Although blood cell numbers begin to recover by 5 dayspost-fertilization (d. p. f), all mutants die between 7-10 d. p. f. Toinvestigate the hematopoietic defect in kgg, we examined the expressionof scl, gata1, and runx1. At the 5-somite stage, the bilateral stripesof scl⁺ cells are thinner in kgg^(tv205) embryos compared to wild-type(wt) controls. In addition, kgg^(tv205) mutants show a decreased numberof gata1⁺ erythroid precursors and a complete absence of runx1expression in blood and neuronal cells. Consistent with the neuronalloss of runx1 expression there are reduced numbers of Rohon-Beard cellsat later stages. By 24 hours post-fertilization (h. p. f.), kgg^(tv205)mutants have a severe reduction in the number of globin-expressingerythroid cells compared to wt siblings. In contrast, normal numbers ofpu.1⁺ myeloid cells^(27, 28) are formed from the cephalic mesoderm inkgg^(tv205) embryos. Similarly, markers of definitive hematopoieticlineages, such as c-myb and rag1, are expressed in kgg^(tv205) mutantsat 36 h. p. f and 6 d. p. f., respectively. To study the development ofthe vasculature in the mutant, we examined the expression of the VEGFreceptor, flk1. At the 10- and 15-somite stages, kgg^(tv205) embryoshave relatively normal numbers of angioblasts, although theirconvergence to the midline is delayed. By 24 h. p. f., the vasculatureappears well formed in the mutants and the few blood cells that developcirculate normally. The pronephric kidney arises from mesoderm adjacentto the ICM precursors²⁹. In kgg^(tv205) mutants, the expression domainsof the pronephric duct markers pax2.1³⁰ and cxcr4b³¹ are shortened,although unlike the scl stripes, the width of the pax2.1 stripe isunaffected. Transcripts for the glomerulus marker wt1²⁹, which arenormally expressed in mesoderm adjacent to somites one through four,extend from somites one through six in kgg^(tv205) embryos suggestingthat the kgg^(tv205) mutation leads to an expansion of anterior kidneyfates at the expense of more posterior fates. Other structures such asthe head, notochord, and somites appear grossly normal in kgg^(tv205)embryos, although the length of the embryo is shortened compared to wtembryos.

Identification of cdx4 as the Gene Defective in kgg Mutants

The kgg mutation maps to linkage group 14 near a number of candidategenes including cdx4³², smad5³³, and wnt8³⁴. An analysis of the cDNAsequence of wnt8 and smad5 from kgg mutants did not identify anymutations. cdx4 belongs to the caudal family of homeobox genes that havebeen implicated in AP patterning³⁵⁻³⁷. Three caudal paralogues exist inmammals (cdx1, cdx2, and cdx4) and mouse gene-targeting studies of cdx1and cdx2 (cdx4 has yet to be targeted) have demonstrated a role forthese genes in the AP patterning of the axial skeleton³⁸⁻⁴⁰. Inaddition, cdx2^(+/−) mice develop hamartomatous polyps in the colon thatresult from a transformation of the intestinal epithelium to a moreanterior (gastric) fate^(39, 41, 42). Sequence analysis of the cdx4 genefrom kgg^(tl240) mutants revealed a T to A transversion in nucleotide+510, changing a conserved F(170) residue in the homeodomain to aleucine. This mutation prevents the protein from binding to a cdx4consensus binding site in gel shift experiments. A partial deletion ofthe cdx4 gene, and at least one other neighboring gene (chic1), wasfound in kgg^(tv205) mutants. To characterize this deletion in moredetail we isolated the cdx4 transcript in kgg^(tv205) mutants by 3′ RACEand found that exon 2 had become spliced onto downstream sequence thatextended the cdx4 open reading frame by 11 amino acids (GFSSVFQSQSD-stop(SEQ ID NO. 31)). Radiation hybrid (RH) mapping of this foreign sequenceplaced it 20 cR away from the cdx4 locus. This analysis confirms thatthe kgg ^(tv205) mutant protein is truncated prior to the homeodomainand indicates that the deletion responsible for the mutation is small(˜0.5 cM). To provide further evidence that the kgg phenotype is causedby defects in cdx4, we injected wt embryos with cdx4 antisensemorpholinos and found that the resulting morphants phenotypicallyresembled kgg embryos.

We next examined the expression pattern of cdx4 during development.Transcripts for cdx4 are first detected in the early gastrula but becomerestricted to the posterior-most cells during gastrulation and earlysomitogenesis. Double whole mount In situ hybridization and sectioningat the 3-somite stage revealed that the cdx4 expression domain initiallyincludes cells in the posterior mesoderm that express scl. However, fromthe 5-somite stage onward the expression domains of cdx4 and scl arelargely non-overlapping. Similar expression profiles were found for themouse orthologues of cdx4 and Scl during early embryogenesis. At thelate primitive streak stage (E7.25), cdx4 transcripts are confined tomesodermal cells of the posterior embryo, the allantois, and the formingyolk sac wall. While cdx4 is not expressed in the nascent blood islands,its expression domain does partially overlap with Scl in mesodermalcells of the posterior primitive streak and the posterior yolk sac.Taken together, these observations are consistent with a conserved,early role for cdx4 during the specification of hematopoietic fate.

Overexpression of cdx4 Induces Ectopic Blood Cells

To further explore the function of cdx4 during embryonic hematopoiesis,we examined the effect of cdx4 overexpression in wt embryos. Embryosinjected with cdx4 mRNA (7, 15, or 30 pg) display a range of“posteriorized” phenotypes. In contrast, embryos injected with 15 pg ofF(170)L mutant mRNA all exhibit a wt morphology (n=60/60 embryosinjected; data not shown). The effect of cdx4 overexpression (15 pg) onblood development was examined at the 5- to 12-somite stages.Surprisingly, 12-20% of the injected embryos showed ectopic scl(n=24/118), gata1 (n=7/59), and fli1 (n=4/26) expression near themidline in a stripe that ran parallel to the endogenous bloodprecursors. Cross sections revealed that the ectopic scl⁺ cells wereunilaterally located adjacent to the notochord. The reason for thisrestricted localization is currently unclear, however the genes inducedappear specific to the hematopoietic program as ectopic flk1 expressionwas confined to the upper trunk region (n=11/69), whereas no ectopicexpression of pax2.1 was found (n=0/55). In contrast, 11-22% of theinjected embryos exhibited decreased expression of scl, gata1, fli1,flk1, and pax2.1. The disrupted tissue development in these embryos mayresult from abnormal gastrulation, or the conversion of mesoderm to anextreme posterior fate. To assess the ability of cdx4 to rescuekgg^(tv205) mutants, we injected 15 pg of cdx4 mRNA and assayed thenumber of scl⁺ and gata1⁺ cells at the 5- and 10-somite stages,respectively. Consistent with cdx4 being the gene defective in kggmutants, the hematopoietic defects were partially rescued inapproximately 80% of injected mutants (n=15/19 mutants for scl andn=27/33 mutants for gata1).

kgg Mutants Have Abnormal hox Gene Expression

In a number of metazoans, caudal homologues have been implicated in APpatterning by regulating the expression of hox genes^(38, 43-45). Toinvestigate hox gene expression in kgg mutants we examined theexpression of selected hoxb cluster genes and hoxa9a, as many of thesehox genes are known to affect haematopoiesis⁵. All of the hox genesexamined (hoxb4, hoxb5a, hoxb6b, hoxb7a, hoxb8a, hoxb8b, and hoxa9a)display altered expression patterns in kgg^(tv205) embryos. Forinstance, the mesodermal expression of hoxb5a normally includes somitestwo and three, the notochord, and the tailbud region, but in kgg^(tv205)mutants, hoxb5a expression is expanded to include somites two to five,is absent from the notochord, and is reduced in the tailbud. In the caseof hoxb6b and hoxa9a, the expression of these hox genes is almost absentin kgg ^(tv205) mutants.

Overexpression of hox Genes Rescues Erythropoiesis in kgg Mutants

To further understand how the stripe of hematopoietic/vascularprecursors is affected by changes in AP patterning, we examined the scl⁺populations in more detail. During normal development, transcripts forscl are first detected around the 3-somite stage in stripes of mesodermadjacent to the future site of somite six. At the 5-somite stage, denovo expression of scl occurs adjacent to somites one to five. Thesecells are most likely angioblasts as they express flk1 but not gata1.Transcripts for flk1 and gata1 in cells of the posterior scl⁺ stripeappear mutually exclusive, suggesting that this stripe is comprised ofjuxtaposed populations of angioblasts and hematopoietic precursors.

In kgg mutants, there is a preferential loss of gata1⁺ hematopoieticcells from the posterior stripe with little effect on the adjacentangioblasts. This blood loss in kgg mutants may result, in part, from aposterior shift in the boundary between the anterior (angioblast) andposterior (blood and angioblast) scl⁺ populations. In support of this,the expression domains of hoxb6b, hoxb7a, and hoxa9a, which share ananterior expression limit with gata1, are significantly reduced inkgg^(tv205) mutants as early as the 3-somite stage. In contrast, thescl⁺ anterior angioblasts are found rostral to the hoxb7a expressiondomain but at a similar AP level as hoxb5a. Given that hox geneoverexpression can transform cell fates¹⁵ and that a number of hox genesare expressed during mouse yolk sac haematopoiesis⁴⁶, we examinedwhether overexpression of hox paralogues from the 6^(th), 7^(th),8^(th), or 9^(th) groups were capable of rescuing the blood defect inkgg^(tv205) mutants. Mutants injected with 3 pg of hoxb7a and hoxa9amRNA displayed an almost complete rescue of gata1⁺ blood cells at the18-somite stage (65%; n=13/20 mutants and 100%; n=18/18, respectively),although the axial and tail defects were not rescued. In contrast, thehighest non-toxic dose of hoxb6b mRNA (1-2 pg; 64%; n=7/11) led to asmall increase in gata1⁺ blood cells, whereas the highest non-toxiclevel of hoxb8a mRNA (1-2 pg) failed to rescue the blood defects (n=0/22mutants; data not shown). Taken together, these findings suggest thatthe specification of hematopoietic cell fate is dependent upon theproper expression of hox genes such hoxb7a and hoxa9a in the posteriormesoderm and that overexpression of any one of these cdx4 targets canrescue erythropoiesis in kgg mutants.

To provide further evidence that cdx4 and hox genes function together ina common pathway, we examined whether cdx4 overexpression (15 pg) couldrescue the expression of hoxb6b, hoxb7a, and hoxa9a in cdx4 morphants.We found a restoration of hoxb6b, hoxb7a, and hoxa9a expression domainsin cdx4-rescued morphants. Interestingly, approximately 80% of theinjected embryos also displayed ectopic hoxb7a expression in theforebrain and/or hindbrain regions (n=31/39), thus supporting a role forcdx4 in the induction of hox gene expression.

Overexpression of scl Fails to Rescue Erythropoiesis in kgg Mutants

In zebrafish, overexpression of scl leads to an expansion ofhematopoietic cells in the posterior lateral plate mesoderm⁸. Weexamined whether scl overexpression could rescue erythropoiesis in kggmutants. Wild-type embryos injected with scl mRNA (100 pg), display anexpanded number of gata1⁺ erythroid precursors at the 10 somite stage.In contrast, no such expansion in erythroid cell numbers was found inscl-injected kgg embryos. Given that cdx4 expression precedes that ofscl in the posterior mesoderm, our results suggest that thespecification of hematopoietic fate by scl is dependent on cdx4.

cdx4 Expands Multipotential Hematopoietic Progenitors Derived fromMurine ES Cells

Several studies have shown that retroviral expression of Hoxb4 inhematopoietic stem cells or multipotential progenitors enhances theself-renewal/proliferation of these cells^(16, 19, 47). To examinewhether cdx4 has a similar activity, we retrovirally transduced embryoidbody (EB) hematopoietic cells with cdx4 and assayed the effect onmultilineage hematopoietic colony formation. In this system, cdx4induced a pronounced expansion of hematopoietic progenitors, including a13-fold increase in CFU-GEMM (colony formingunit-granulocyte/erythroid/macrophage/megakaryocyte) colonies and a11-fold increase in CFU-GM colonies compared to GFP-only transducedcontrol cells (FIG. 1). The cdx4-mediated expansion of multilineageprogenitors and colony size was more potent than that observed withHoxb4, which induced a 9-fold increase in CFU-GEMM (FIG. 1). We nextexamined changes in the expression of selected HoxA, HoxB, and HoxCcluster genes in the cdx4-transduced cells using quantitative PCR.Consistent with the role of cdx4 as a Hox gene regulator, we foundwidespread alterations in Hox expression levels in cells transduced withcdx4 compared to controls (FIG. 2). Notably, cdx4 induced a markedincrease in the expression of HoxB4 (30-fold), HoxB3 (19-fold), HoxB8(5-fold) and HoxA9 (4.1-fold), all of which have been implicated inhematopoietic stem cell or immature progenitor expansion^(18, 48, 49).Taken together, these results suggest that cdx4 can enhance theproliferation of early hematopoietic progenitors by up-regulating theexpression of target Hox genes.

In EBs, precursors committed to primitive and definitive hematopoieticfates arise between day 3 and 4 of differentiation⁵⁰. Consistent withour expression analyses in vivo, we find endogenous expression of cdx4at day 3 and 4 of EB development (FIG. 3). To more closely investigatethe time window during EB differentiation in which cdx4 can enhancemultilineage hematopoietic colony formation we engineered ES cells toexpress cdx4 under the control of a tetracycline-inducible promoter. A‘pulse’ of cdx4 expression was induced at different intervals during EBdifferentiation and hematopoietic colony formation was assayed at day 6(FIG. 4). The strongest effect of cdx4 overexpression on colonyformation was found between day 4 and 5 of EB development with increasedmultipotent progenitors (CFU-GEMM), CFU-GM, and primitive erythroidcolonies compared to uninduced EBs (FIG. 4). These findings areconsistent with cdx4 acting at early stages of hematopoietic developmentto expand the number of multipotential progenitor cells and perhaps,hematopoietic stem cells.

Discussion

Our studies demonstrate that cdx4 is essential for hematopoieticdevelopment during vertebrate embryogenesis. Defects in cdx4 lead to anearly deficit in scl-expressing hematopoietic precursors, whereasoverexpression of cdx4 in zebrafish embryos or mouse ES cells inducesblood formation. Loss of cdx4 function is also associated withwidespread perturbations in the expression patterns of multiple hoxgenes. Furthermore, ectopic expression of cdx4 in both zebrafish andmouse cells alters hox gene expression. The rescue of blood developmentin kgg mutants by overexpressing specific hox genes suggests a pathwayin which cdx4 acts upstream of the hox genes to control embryonic blooddevelopment.

Genetic studies in Drosophila led to the proposal that hox genesfunction in specific combinations to confer tissue identities along theAP axis^(1, 2). In kgg mutants, the expression domains of hox genesexpressed in the anterior trunk, such as hoxb4 and hoxb5a, are expandedtowards the posterior while others such as hoxb6b, hoxb7a and hoxa9a areseverely reduced. With regard to the development of ICM precursors,these perturbations in hox expression domains appear to cause aposterior shift in the boundary between the anterior endothelialpopulation and the more posterior populations of blood and endothelialcells. In addition, there is an overall reduction in erythroid cellnumbers (schematically represented in FIG. 5). The blood defects in kggmutants can be restored to almost wild-type levels by overexpressinghoxa9a and hoxb7a, whereas hoxb6b rescues poorly and hoxb8a fails torescue. These observations suggest that multiple hox genes withredundant activities participate in blood development. In support ofthis redundancy, the targeted disruption of HoxB6, HoxB7, or HoxA9 inmice does not block early embryonic haematopoiesis⁵¹⁻⁵³. Similarly,using morpholinos to knock-down multiple hox genes we have been unableto find single or combinations of hox genes that are required for bloodformation during zebrafish development. However, there are technicallimitations to this approach as non-specific toxicity makes it difficultto inject more than three morpholinos simultaneously.

Our finding that scl overexpression fails to rescue blood development inkgg mutants suggests that the cdx4-hox pathway may be required to makethe posterior lateral plate mesoderm competent to respond to factorsthat specify hematopoietic fate. In addition to scl, these factors arelikely to include other molecules such as BMPs, as we have found thatenhancing BMP signaling also fails to rescue the blood defect in kggmutants. A role for hox genes as ‘competence’ factors during blooddevelopment may explain the restricted localization of ectopic bloodcells induced by cdx4 overexpression. Rather than being distributedthroughout the embryo, the ectopic blood forms a stripe near the midlinethat is parallel to the endogenous stripes of hematopoietic precursors.The parallel nature of the cdx4-induced blood cells suggests that thegenes responsible for patterning the endogenous stripes may also beresponsible for restricting the localization of the ectopic blood. Inthis model, cdx4 overexpression would induce a combination of hox genesthat renders the injected cells competent to respond to other pathwaysacting upstream of scl. The spatial localization of these signals andthe influence of other patterning factors would then account for therestricted stripe of cdx4-induced blood.

Our results have implications for the concept of the hemangioblast, aputative bipotential cell that is thought to express scl and give riseto both blood and vascular lineages in vivo⁵⁴. kgg mutants display areduced number of scl⁺ cells with a selective loss of blood but notangioblasts. This finding suggests that if hemangioblasts exist in vivothen they must arise prior to the onset of scl expression and that cdx4is necessary for this putative population to differentiate into an scl⁺hematopoietic precursor. Alternatively, the blood and vascular lineagesmay arise independently from the posterior mesoderm with cdx4 beingrequired solely for the specification of hematopoietic fate. Eithermodel does not rule out the possibility that early scl⁺ cells stillretain the plasticity to form both blood and vascular lineages iftransplanted or cultured in a suitable environment.

Our experiments support a conserved role for cdx4 in the formation ofhematopoietic cells during vertebrate embryogenesis. Like the zebrafishorthologue, mouse cdx4 expression overlaps with scl in posterior regionsof the conceptus. In addition, cdx4 transcripts are enriched in theRhodamine-123 low fraction of adult mouse bone marrow, which containsthe long term repopulating stem cell (Thor Lemischka pers. comm.).Overexpression of cdx4 in EBs promotes the formation of multilineageprogenitors and alters the expression of multiple Hox genes. Theinduction of hematopoietic progenitors by cdx4 is similar to that seenwith HoxB4 overexpression. Furthermore, cdx4 is able to upregulate theexpression of HoxB4 in EBs, raising the possibility that HoxB4 mediatesthe effect of cdx4 on multilineage progenitor expansion. Given thatHoxB4 can also confer upon primitive progenitors the ability to engraftlethally irradiated adults²⁰, it will be interesting to examine thelong-term, multilineage potential of cdx4-expressing progenitors in thisassay. Unlike HoxB4, overexpression of cdx4 in EBs leads tosignificantly more CFU-GM colonies compared to the control. Thisdifference may result from other Hox genes, or combinations of Hoxgenes, that are induced by cdx4.

Deregulated expression of Hox genes by retroviral activation,chromosomal translocation, or upregulation as a result of mutations inupstream activators have all been implicated in leukemictransformation⁵. The function of cdx genes as transcriptional regulatorsof hox genes raises the possibility that this family may alsoparticipate in leukemogenesis. In support of this, a fusion of CDX2 toTEL/ETV6, a gene frequently rearranged in hematological malignancies,has been found in a patient with acute myeloid leukaemia⁵⁵. CDX2expression, which is not normally found in hematopoietic cells, was alsoobserved in a case of leukemia lacking the translocation, suggestingthat ectopic expression of CDX2 can also occur by other mechanisms⁵⁵.The challenge for future studies will be to better understand how hoxgenes downstream of cdx genes regulate commitment to a hematopoieticfate and participate in leukemia.

Example 2

Injection of cdx2 morpholinos into kugelig/cdx4 mutants (resulting incdx2 and cdx4 deficient embryos) results in a complete absence of gata1⁺erythroid precursors and a more severe shortening of the embryonic axisat the 10 somite stage. In contrast, vascular progenitors and kidneyduct precursors appear to be little, or unaffected, compared to kggsingle mutants. Embryos deficient in just cdx2 display normal blooddevelopment. Expression of cdx2 and cdx4 overlaps during gastrulationand early somite formation at the time that hematopoietic cells ariseduring embryogenesis. Taken together, these findings suggest that cdx2and cdx4 act redundantly during development to control the formation ofblood cells.

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The present invention has been described in terms of particularembodiments found or proposed by the present inventor to comprisepreferred modes for the practice of the invention. It will beappreciated by those of skill in the art that, in light of the presentdisclosure, numerous modifications and changes can be made in theparticular embodiments exemplified without departing from the intendedscope of the invention. For example, due to codon redundancy, changescan be made in the underlying DNA sequence without affecting the proteinsequence. Moreover, due to biological functional equivalencyconsiderations, changes can be made in protein structure withoutaffecting the biological action in kind or amount. All suchmodifications are intended to be included within the scope of theappended claims.

1. A method for enhancing proliferation or hematopoietic differentiationof a mammalian stem cell comprising, transfecting said stem cells in anin vitro culture medium with an exogenous nucleic acid comprising a cdxcoding sequence operably linked to a promoter.
 2. The method of claim 1,wherein the stem cell is a hematopoietic stem cell.
 3. The method ofclaim 1, wherein the cell is a CD34⁺ cell.
 4. The method of claim 1,wherein the cell is autologous.
 5. The method of claim 1, wherein thecell is obtained from a human.
 6. The method of claim 5, wherein thehuman is suffering from, or is susceptible to, decreased blood celllevels.
 7. The method of claim 6, wherein the decreased blood celllevels are caused by chemotherapy, radiation therapy, bone marrowtransplantation therapy or congenital anemia.
 8. The method of claim 1,wherein the exogenous nucleic acid is a retroviral vector.
 9. The methodof claim 1, wherein the exogenous nucleic acid is an episomal vector.10. The method of claim 1, wherein the stem cell is an embryonic stemcell.
 11. The method of claim 1, wherein the cdx is selected from thegroup consisting of cdx 1 and cdx
 2. 12. A method of treating a mammalin need of improved hematopoietic capability, comprising the steps of:(a) removing hematopoietic stem cells from the mammal; (b) transfectingsaid stem cells with exogenous nucleic acid comprising cdx sequences;(c) culturing said transfected stem cells to form an expanded populationof stem cells; and (d) returning said expanded cells to the mammal,whereby hematopoietic capability is improved.
 13. The method of claim12, wherein the mammal is a human.
 14. The method of claim 12, whereinthe exogenous nucleic acid is a retroviral vector.
 15. The method ofclaim 12, wherein the cdx is selected from the group consisting of cdx 1and cdx
 2. 16. A method for enhancing proliferation or hematopoieticdifferentiation of a mammalian stem cell comprising, treating said stemcells by addition in an in vitro culture medium of an exogenous cdxpeptide.
 17. The method of claim 16, wherein the stem cell is ahematopoietic stem cell.
 18. The method of claim 16, wherein the cell isa CD34⁺ cell.
 19. The method of claim 16, wherein the cell isautologous.
 20. The method of claim 16, wherein the cell is obtainedfrom a human.
 21. The method of claim 20, wherein the human is sufferingfrom, or is susceptible to, decreased blood cell levels.
 22. The methodof claim 21, wherein the decreased blood cell levels are caused bychemotherapy, radiation therapy, bone marrow transplantation therapy, orcongenital anemia.
 23. The method of claim 16, wherein the stem cell isan embryonic stem cell.
 24. The method of claim 16, wherein said cdx isgenetically fused to a transport moiety.
 25. The method of claim 24,wherein said transport moiety is a fragment of HIV tat protein.
 26. Themethod of claim 16, wherein the cdx is selected from the groupconsisting of cdx 1 and cdx
 2. 27. A method of treating a mammal in needof improved hematopoietic capability, comprising the steps of: (a)removing hematopoietic stem cells from the mammal; (b) treating saidstem cells by administration of exogenous cdx4 peptide; (c) culturingsaid stem cells to form an expanded population of stem cells; and (d)returning said expanded cells to the mammal, whereby hematopoieticcapability is improved.
 28. The method of claim 27, wherein the mammalis a human.
 29. The method of claim 27, wherein the cdx is selected fromthe group consisting of cdx 1 and cdx 2.